Antiquorum Sensing Potential of Andrographolide from Andrographis paniculata in Vibrio harveyi

 

R. Nancy Immaculate Mary1*, N. Banu2

1Research Scholar, Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies, Chennai

2Associate Professor, Department of Biotechnology, Vels Institute of Science, Technology and Advanced Studies, Chennai.

*Corresponding Author E-mail:

 

ABSTRACT:

The recently discovered cell–cell communication system through signaling molecules among bacterial pathogens is known as Quorum sensing. It is well known for bacterial virulence, antibiotic resistance and biofilm production. Microbial pathogenicity is reduced by inhibiting the quorum sensing activity in all infections caused by bacteria. In this present study the compound Andrographolide which is present as a secondary metabolite in Andrographis paniculata was assessed for its anti quorum sensing activity against acyl homoserine lactone (AHL)-dependent biofilm formation and bioluminescence production in Vibrio harveyi (MTCC 7771). The Andrographolide showed a concentration dependent (100 μg/ml) reduction in biofilm production to a level of 97% and reduction of bioluminescence at the level of 98% in Vibrio harveyi (MTCC 7771) and compared with the standard Andrographolide (sigma aldrich). Light microscopic analysis and  Microtiter plate assay further confirmed the reduction in the biofilm forming ability of Vibrio harveyi (MTCC 7771) when treated with andrographolide. The andrographolide at its standard concentration showed antibacterial effect on Vibrio harveyi(MTCC 7771).

 

KEYWORDS: Andrographolide, Andrographis paniculata, Vibrio harveyi, Bioluminescence, Microtiter plate assay.

 

 


INTRODUCTION:

Vibrio harveyi is a Gram-negative, bioluminescent, marine bacterium in the genus Vibrio. Vibrio harveyi is rod-shaped, motile (via polarflagella), facultatively anaerobic, halophilic, and competent for both fermentative and respiratory metabolism. It does not grow below 4 C or above 35 C. V. harveyi can be found free-swimming in tropical marine waters, commensally in the gut micro flora of marine animals, and as both a primary and opportunistic pathogen of marine animals, including Gorgonian corals, oysters, prawns, lobsters, the common snook, barramundi, turbot, milkfish, and seahorses[1].

 

It is responsible for luminous vibriosis, a disease that affects commercially farmed penaeid prawns.[2] Additionally, based on samples taken by ocean-going ships, Vibrio harveyi is thought to be the cause of the milky seas effect, in which, during the night, a uniform blue glow is emitted from the seawater. Some glows can cover nearly 6,000 sq m (16,000 km2). Groups of Vibrio harveyi bacteria communicate by quorum sensing to coordinate the production of bioluminescence and virulence factors. Quorum sensing was first studied in V. fischeri (now Aliivibrio fischeri), a marine bacterium that uses a synthase (LuxI) to produce a species specific  autoinducer (AI) that binds a cognate receptor (LuxR) that regulates changes in expression. Coined "LuxI/R" quorum sensing, these systems have been identified in many other species of Gram-negative bacteria.[3] Despite its relatedness to Vibrio fischeri, Vibrio harveyi lacks a LuxI/R quorum-sensing system, and instead employs a hybrid quorum-sensing circuit, detecting its AI through a membrane-bound histidine kinase and using a phosphorelay to convert information about the population size to changes in gene expression.[4] Since their identification in Vibrio harveyi, such hybrid systems have been identified in many other bacterial species. Vibrio harveyi uses a second AI, termed autoinducer-2or AI-2, which is unusual because it is made and detected by a variety of different bacteria, both Gram-negative and Gram-positive[5,6,7]. Thus, Vibrio harvey has been instrumental to the understanding and appreciation of interspecies bacterial communication. Andrographis paniculata, (Burm. F.)  Wall. ExNees, a herbaceous plant belonging to the Family Acanthaceae, is one of the most commonly used medicinal plants in the traditional systems of Unani and Ayurvedic medicines. It grows in hedge rows throughout the plains of India and is also cultivated in gardens. It also grows in many other Asian countries and is used as a traditional herbal medicine in China, Hong Kong, the Philippines, Malaysia, Indonesia, and Thailand. It is an annual plant of 1-3 ft high, also known as the “king of bitters”, being the aerial parts most commonly used. Andrographolide paniculata have shown a broad range of pharmacological effects such as inhibition of replication of the HIV virus, prevention of common cold, and antimalarial, antidiarrheal, antibacterial, antihyperglycemic effects, suppression of various cancer cells, and principally anti-inflammatory properties. Andrographolide is the major labdanediterpenoid isolated from Andrographolide paniculata and exhibits anti-inflammatory and anticancer activities, either in vitro or in vivo experimental models of inflammation and cancer.

 

MATERIALS AND METHODS:

Preparation of Andrographis paniculata leaf Extracts:

Andrographis paniculata leaves used in this study were collected from well grown trees in Tamilnadu Agriculture University, Katuthotam, Thanjavur district, Tamilnadu. The leaves were washed twice with ordinary water followed by distilled water [13]. The washed leaves were shade dried and powered using blender. 5gm of powered sample were soaked in 50 ml of methanol for overnight. The methanol phase was collected and dried at 55°C. The residues were collected and redissolved with deionized water. Finally stored at -20°C for further use.

 

Bacterial Strains and Their Culture Conditions:

Vibrio harveyi (MTCC 7771) used in this study is obtained from MTCC (The Microbial Type Culture Collection and Genebank), Chennai. The bacterial strain was allowed to grow aerobically in Luria– Bertani slants. From the slants, sub culture were made and cultures were maintained in (LB) broth (Hi-Media, India) at an optimum temperature (30° C) and intensity was checked using UV spectrometer at OD 600nm before using for further experiments.

 

Micro Titer Plate Assay:

The effect of Andrographis paniculata extract on the biofilm formation of bacterial pathogens was determined by quantifying the biofilm biomass through MTP assay11. Briefly, 1% of overnight cultures (OD adjusted to 0.4 at 600 nm) of test pathogens were added into 1 ml of fresh Luria Bertani (LB) medium and cultivated in the presence and absence of Andrographis paniculata extract (20,50,75,100 µg/ml) without agitation for 16 h at 30 °C. After incubation, the planktonic cells in MTPs were removed by rinsing the wells twice with sterile water. The surface-adhered cells in the MTP wells were stained with 200 μl of 0.2% crystal violet (CV) solution (Hi Media, India). After 10min, excess (CV) solution was removed and CV in the stained cells was solubilized with 1 ml of 95% ethanol. The bio film biomass was quantified by measuring the intensity of CV at OD 650 nm using UV–visible spectrophotometer [12].

 

Growth Curve Analysis:

The Vibrio harveyi culture at the concentration of  one percentage (0.4 OD at 600 nm) were inoculated in 250 ml Erlenmeyer flask containing 100 ml of LB broth supplemented with various concentrations (20,50,75,100 μg) of Andrographis paniculata extract. The flasks were incubated at the optimum temperature which is suitable for Vibrio harveyi under 180 rpm in a rotatory shaker. The cell density was measured in UV– visible spectrophotometer at every one hour interval [4].

 

Light Microscopic Analyses:

Briefly, 1% of overnight cultures of the test pathogens (0.4 OD at 600 nm) were added into 1 ml of fresh LB medium containing cover glass of 1 cm2 along with and without Andrographis paniculata extracts (20, 50, 75, 100µg/ ml). After 16 h of incubation, the cover glasses were rinsed thrice with distilled water to remove the plank tonic cells [10]. The bio films in the cover glasses were stained with 0.2% CV solution. Stained cover glasses were placed on slides with the bio film pointing up and visible bio films were visualized by light microscope at magnifications of 40X (Olympus CK ×41 Jenoptik Germany, Pro Res C5).

 

Bioluminescence Assay:

One percentage of V. harveyi cells at the aforesaid cell density were inoculated into 5 ml of alkaline peptone water (APW) and cultivated in the presence and absence of Andrographis paniculata at various concentrations (20,50,75,100 µg/ml) and then incubated at 30 °C for 12 h. After incubation, the intensity of bioluminescence was measured in terms of relative light units (RLU) using a luminometer. The percentage inhibition of bioluminescence was calculated based on the obtained RLU [8].

 

Antibacterial assay:

Antibacterial activity of the Andrographis paniculata leaf extracts with QSI potential was performed in Muller–Hinton agar (MHA) (Hi Media, India) by the method followed by the Clinical and Laboratory Standards Institute (2006). The 100 μl of test bacterial suspensions which were expected to have cell density equivalent to 0.5 McFarland standards (approximately 1×108 CFU/ml) were uniformly spread over the surface of the MHA plate [9]. Then, the sterile paper disks (Hi Media, India) with a diameter of 10 mm loaded with various concentrations.

 

RESULTS:

Bio film Control and Light Microscopic Analysis:

In the present study, bio film images revealed that the Andrographolide an Andrographis paniculata extracts effectively disturb the bio film formation as shown in light microscopic analysis. In this study, the bacterial cultures were used as target pathogenic model to knowanti-bio film activity of Andrographis paniculata . The minimum inhibition of bio film was 20µg/ml and maximum inhibition of bio film were 100µg/ml (Fig: 1). In order to analyze the anti bio film efficiency of Andrographis paniculata , extract in inhibiting bio film formation, Pseudomonas aeruginosa cells were allowed to grow in MTP having glass slide in the presence and absence of Andrographis paniculata extract and the results were identified and visualized under alight microscope (Fig: 2).

 

 Fig: 1. Light microscopic view of Vibrio harveyi treated with andrographolide extracted from with Andrographis paniculata

 

As bioluminescence in V. harveyi is positively controlled by Andrographolide collected from ethanolic leaf extract of Andrographis paniculata, the anti- QS potential of Andrographis paniculata was assessed using V. harveyi. As expected, Andrographis paniculata extract (20, 50, 75, 100µg/ml) exhibited a dose-dependent inhibition in bioluminescence and a maximum of 98% inhibition of bioluminescence with 100µg/ml (Fig: 3).


 

Fig: 2 Inhibition of Biofilm by Andrographolide in Vibrio harveyi

 

Fig: 3 Inhibition of Bioluminescence in Vibrio harveyi by Andrographolide

 


 

CONCLUSION:

In this study,the compound Andrographolide was collected from the methanolic leaves extract of  Andrographis paniculata, and the possibility of anti-bio film activity was been ruled out. Therefore, it is found that Andrographolide is acting as an active principle exhibiting QSI activity might be an analogue of AHL molecule. However, the extract needs further purification and characterization and compared with the standard to confirm the inhibitor activity. In conclusion, the reduction in QS and the end effect on virulence factors production without affecting the bacterial growth provided some insight into the potential of Andrographolideas QS inhibitor. Therefore, it is concluded that the QSI potential of these Andrographolide could be used as anti-bio film drug to combat with bacterial infections.

 

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Received on 05.12.2016             Modified on 27.12.2016

Accepted on 14.01.2017           © RJPT All right reserved

Research J. Pharm. and Tech. 2017; 10(2): 449-452.

DOI: 10.5958/0974-360X.2017.00090.7